Regulation of the activity of sodium, potassium-ATPase (NKA, the sodium pump) is a critical element in the maintenance of extracellular fluid volume and blood pressure. When the enzyme is resident in its functional location in the cell membrane, regulation of the enzyme is possible at both the intracellular and extracellular surfaces. At the intracellular surface the enzyme is regulated by signals arising from cell surface receptors for humoral substances such as angiotensin II, dopamine and norepinephrine. There is now evidence that these signals alter the vectorial transport produced by NKA by changing its subcellular distribution (endosomal sequestration). At its extracellular surface, NKA projects a highly conserved cardiotonic steroid binding site which is an integral part of the enzyme and which serves no other function except to bind cardiotonic steroids with high affinity. Recent work in our laboratory and work by others in the field has defined the chemical identity of substances which bind to and inhibit the enzyme through the cardiotonic steroid receptor. We have shown that this inhibitor is an endogenous substance synthesized by the adrenal cortex. The present studies will further refine our understanding of the function of adrenocortical cardiotonic steroid. The present studies will further refine our understanding of the function of adrenocortical cardiotonic steroid. We will delineate important features of the biosynthetic pathway which leads to formation of the endogenous adrenal cardiotonic steroid. We will examine the role of cholesterol and cholesterol side chain cleavage in the metabolic pathway leading to the production of adrenal cardiotonic steroid. We will examine the control of activity in this novel steroidogenic surface receptors. Finally, we will examine and characterize the interaction between adrenal cardiotonic steroid and its receptor binding site on renal sodium, potassium-ATPase. We will also determine if the interaction with this site coordinates with regulation of ATPase activity by humoral substances and whether the mechanism of coordination occurs through increased sequestration of the receptor protein.